Antibody reagents that identify the carboxy-terminal peptide of the GTP-binding protein Go

ABSTRACT

Antibodies having binding specificity for a peptide having the sequence ANNLRGCGLY have been prepared. The antibodies bind to the GTP-binding protein G o  and are useful for identifying, isolating and distinguishing between different GTP binding proteins.

This application is a divisional of application Ser. No. 07/654,675,filed on Aug. 8, 1990 and now abandoned, which was a continuationapplication of Ser. No. 07/365,919 filed on Jun. 15 1989, now abandoned,which was a divisional of Ser. No. 07/100,909 filed on Sep. 25, 1987,now U.S. Pat. No. 4,870,161.

BACKGROUND OF THE INVENTION

The present invention is related to guanine nucleotide-binding proteins.More particularly, the present invention is related to specific reagentsand probes for identifying, isolating and distinguishing betweendifferent guanine nucleotide (GTP)-binding proteins.

STATE OF THE ART

GTP-binding (hereinafter "G" proteins) comprise a family of distinct butrelated signal transducers. One or more members of the family is foundin virtually every type of cell, and performs a critical role in thetransduction of hormonal, neurotransmitter, cytokine, odorant, and lightsignal functions. G-proteins are known to be heteromeric; thealpha-GTP-binding subunit is distinct for each member of the G-proteinfamily and is believed to confer specificity in both receptor andeffector interactions.

A combination of protein purification and recombinant DNA techniqueshave led to the realization that the G-protein family consists of atleast 7 distinct members. Cloning of complementary DNA for each of theseallows prediction of the amino acid sequence. The proteins include 2forms of "transducin" (TD), a G-protein uniquely found in retinalphotoreceptors and involved in phototransduction, and five otherproteins termed G_(s), G_(o), G_(i1), G_(i2), and G_(i3). The specificfunctions of each of these is as yet unclear, but they appear toregulate ion channels and enzymes that generate intracellular "secondmessengers." Although each protein shows significant amino acid sequencehomology (ranging from 40% for G_(s) vs. G_(i), to 90% for G_(i1) vs.G_(i3)), there are unique sequence differences in each.

Certain bacterial toxins, particularly pertussis toxin, covalentlymodify certain G-proteins and this technique has been used foridentification of G-proteins. However, this method is relativelynonspecific as transducin (TD), G_(o), and all forms of G_(i) appear tobe pertussis toxin substrates. For this reason, several laboratorieshave generated antibodies against G-proteins to obtain more specificprobes. The majority of these reagents, whether polyclonal rabbitantisera or monoclonal mouse antibodies are of undefined epitope, andare not particularly specific for a single G-protein so as todefinitively distinguish one from the other. Moreover, none of the knownantibodies can be used to block specific functional interactions ofG-proteins.

SUMMARY OF THE INVENTION

It, is, therefore, an object of the present invention to providesynthetic peptides corresponding to specifically defined sequences ofG-protein alpha subunits.

It is another object of the present invention to provide antisera orpurified antibodies having binding affinity exclusively for a particularepitopic site of a G-protein.

It is a further object of the present invention to provide a reagent kitfor identifying or distinguishing between specific G-proteins.

It is yet another object of the present invention to provide Uniqueprobes for isolating specific G-protein antibodies in substantially pureform.

Other object and advantages of the present invention will become evidentfrom the Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered connection with theaccompanying drawings wherein:

FIG. 1 shows the detection of Gi-alpha mono-ADP-ribosylated by pertussistoxin with AS/7. C6 glioma cells were treated in culture for 16 hourswith: a) no toxin, b) 100 ng/ml pertussis toxin, c) 100 ng/ml choleratoxin. A crude plasma membrane fraction was then prepared and 50 μg/lanemembrane protein were separated by SDS-PAGE on a 10% gel. Immunoblotswere performed as described in the specification, except that a 1:500dilution of second antibody was used, and O-diansidine (0.025% solution)was used as substrate for the enzyme-2nd antibody conjugate. A 1:100dilution of AS/7 was used as first antibody. The positions of molecularweight standards are indicated;

FIG. 2 shows the detection of brain "Gi-alpha" with AS/6 and 7. 100μg/lane of cholate extract of bovine cerebral cortical membranes wereseparated by SDS-PAGE on a 10% gel and immunoblotted, except thatsamples were treated with N-ethylmaleimide before SDS-PAGE. Antiseraused were: affinity purified RV/3 (FIGS. 2A and B) at lanes 1 and2--1:20 dilution, and lane 3--1:40 dilution; AS/6 (FIG. 2A) and AS/7(FIG. 2B) at lane 2--1:40, lane 3--1:200 dilution, and lane 4--1:00dilution. The positions of alpha subunits of Gi and Go and of the commonbeta subunit are indicated;

FIG. 3 shows the detection of "Gi-alpha" in brain and neutrophil withantisera LE/1,2 and 3 and AS/6 and 7. (In FIG. 3A, 50 μg/lane of highlypurified human neutrophil plasma membranes, and in FIG. 3B, 150 μg ofbovine brain membrane cholate extract were separated by SDS-PAGE on a10% gel and immunoblotting performed). Antisera used were: preimmuneLE/1 (lane 1), immune LE/1 (lane 2), preimmune LE/2 (lane 3), immuneLE/2 (lane 4), preimmune LE/3 (lane 5), immune LE/3 (lane 6), immuneAS/6 (lane 7), and immune AS/7 (lane 8). LE/1, 2 and 3 (immune andpreimmune) were used at 1:100 dilution, and AS/6 and 7 were used at1:250 dilution. The position of the Gi-alpha subunit is indicated; and

FIG. 4 shows the reactivity of antisera with purified GTP-bindingproteins. The major pertussis toxin substrate (.alpha subunit only)purified from bovine neutrophils (lane 1), Gi/Go purified from bovinebrain (lane 2), and holotransducin (lane 3) purified from bovine rodouter segments were separated by SDS-PAGE on a 10% gel. In FIG. 4A, thegel was stained for protein, and in FIG. 4B immunoblotting was performedwith the indicated antisera (LE/3 and CW/6, 1:100 dilution; AS/6, 1:250dilution). In lane 1 FIG. 4A 0.5 μg was loaded, in the lane 1 FIG. 4B1.0 μg was loaded. In lane 2 (A and B), 4 μg were loaded, and in lane 3(A and B) 2 μg were loaded. The positions of alpha and beta subunits areindicated.

DETAILED DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the presentinvention are achieved by providing antisera or isolated, substantiallypure antibodies having binding affinity for specific synthetic epitopeof G-protein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

The term "substantially" pure as used herein means that the preparationis as pure as can be obtained employing standard techniques well knownto one of ordinary skill in the art.

MATERIALS AND METHODS

Peptide synthesis: The synthetic peptides were assembled stepwise by theMerrifield solid-phase method (Barany et al, The Peptides 2A:1-284,1979), using an Applied Biosystems 430A automated peptide synthesizer.The peptide resins were cleaved with anhydrous hydrogen fluoride and thecrude peptides were purified by preparative liquid chromatography onreverse-phase C18. The purified peptides were homogeneous by analyticalhigh-performance liquid chromatography, and gave amino acid compositionsconsistent with those theoretically expected. Table 1 lists the aminoacid sequences of the synthetic peptides and the designation of therabbit antisera produced therefrom.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Sequence Of                                                                          Designation Of                                              Synthetic Peptides                                                                              The Antisera                                                (Single Letter Code)                                                                            vs Synthetic Peptide                                        ______________________________________                                        KENLKDCGLF        AS/6, AS/7                                                  LERIAQSDYI        LE/2, L3/3                                                  LDRIAQPNYI        LD/1, LD/2                                                  ANNLRGCGLY        GO/1, GO/3                                                  GCTLSAEERAALERSK  GC/1, GC/2                                                  ______________________________________                                    

Peptide Conjugation and Immunization: 10 mg of keyhole limpet hemocyanin(KLH, Sigma) and 3 mg of peptide were dissolved in 1.0 ml of 0.1Mphosphate buffer, pH 7.0, 0.5 ml of 21 mM glutaraldehyde (also in 0.1Mphosphate buffer, pH 7.0) was then added dropwise with stirring and thecombined 1.5 ml were incubated for about 24 hours at room temperature(about 22° C.-25° C.). The 1.5 ml solution was mixed with an equalvolume of complete Freund's adjuvant and 1 ml aliquots of the resultingemulsion were injected in multiple intradermal sites in 3 New Zealandwhite rabbits. Four weeks later each animal received a boosterimmunization with material prepared indentically except that 1/2 as muchpeptide and KLH were injected in incomplete Freund's adjuvant. Preimmunesera were collected, and subsequent bleeds were performed weekly,beginning two weeks after booster immunization. Affinity-purification ofantibodies from the antisera was accomplished by standard techniquesusing immoblized holoTD as described for example by Gierschik et al,1986 (Proc. Natl. Acad. Sci USA 83:2258-2262). The purified antibodiesare conveniently cryopreserved in suitable buffers.

Membrane Preparations: Human neutrophils were isolated and plasmamembrane-enriched fractions prepared as described by Falloon et al,(FEBS Letters 209:3520356, 1986). C6 glioma cells were cultured andmembrane preparations made as described by Milligan et al, (FEBS Letters195:225-230, 1986). Bovine brain membrane fractions and cholate extractswere made as described by Sternweis et al (J. Biol. Chem.259,13806-13813, 1984) and Gierschik et al (supra).

Protein Purification: Synthetic peptides of the present invention wereemployed as probes for affinity purification of specific antibodiesfollowing standard methodology well known in the art. TD was purifiedfrom bovine rod outer segment membranes as described by Gierschik et al.(Proc. Natl. Acad. Sci. USA 82:727-731, 1985). A mixture of Gi and Gowas purified from bovine brain as described by Milligan et al, (J. Biol.Chem. 260:2057-2063, 1985) and Gierschik et al, 1986 (supra.). The "48k"protein was purified from bovine retinas as described by Zigler et al,(Invest Ophthalmol. Vis. Sci. 25:977-980. 1984). The major pertussistoxin substrate of bovine neutrophils was purified as the isolated alphasubunit.

Other methods: SDS-PAGE and immunoblotting were performed as describedby Gierschik et al, (supra. 1985) and Gierschik et al, (J. Biol. Chem.261:8058-8062, 1986). Incubation with first antibody solutions were forabout 24 hours at room temperature. Dilutions of antisera used in firstantibody solutions have been indicated, supra. Second antibody,peroxidase-conjugated goat anti-rabbit Ig (Kierkegarrd and Perry), wasused at a 1:300 dilution, and incubation was for about 2 hours at roomtemperature. 4-chloronaphthol (Sigma) was used as substrate forperoxidase. Pertussis toxin (List Biochemicals) was activated and usedin ADP-ribosylation reactions with either nonradioactive NAD (1 mM) or³² P-alpha-NAD (10 μM) as described by Eide et al, (Biochemistry25:6711-6715, 1986).

FIGS. 1-4 present data showing specificity of various antisera orantibody reagents of the present invention with respect to variousG-proteins. A summary of the results obtained is presented in Table 2.It should be noted that the cascade of the antibody reagent of thepresent invention for the first time enables identification anddifferentiation between various members of the G-protein familyincluding subtyping of new ones. In addition, the availability of theisolated, purified antibodies allow the determination of the functionalrole of specific G-proteins by interacting receptor or effector siteswith the antibodies or a fragment or conjugate thereof. Technique usedfor such studies are well known to one of ordinary skill in the art towhich this invention belongs.

                  TABLE 2                                                         ______________________________________                                        Specificity of Various Rabbit Antisera to Particular G-Proteins               Antisera     Specificity                                                      ______________________________________                                        AS/6 AS/7    Specific for TD (both forms) and                                              for G.sub.i1,2,3 but not for G.sub.o.                                         Blocks receptor-G-protein coupling.                              LE/2 LE/3    Specific for G.sub.i2 only.                                      LD/1 LD/2    Specific for G.sub.i1, and not G.sub.o or                                     G.sub.i2.                                                        GO/1 GO/3    Specific for G.sub.o only. Blocks                                             receptor-G-protein coupling.                                     GC/1 GC/2    Reacts primarily with G.sub.o ; to                                            lesser extent with G.sub.i forms.                                ______________________________________                                    

In contrast, the antisera or antibody reagents heretofore known eithercompletely lack or have limited specificity for particular G-proteins soas to allow distinction between them. Table 3 comparatively lists thepoperties of prior art antibodies. It should be noted that the prior artantibodies were in general prepared by employing the entire (holo)protein and not by using specific epitopes, identified and synthesizedas in the present invention. Hence, the prior art antibody reagents lackspecificity, i.e. for such G-proteins as G_(i1),2,3 OR G_(o) and thelike. Thus, differentiation between the various subtypes of theG-protein family is simply not possible with heretofore availableantibody reagents.

                  TABLE 3                                                         ______________________________________                                        Reference Type Ab   Specificity                                                                              Antigen Used                                   ______________________________________                                        Gierschik polyclonal                                                                              TD         Raised vs. pure                                et al PNAS                     holo-protein                                   82:727, 1985                                                                  Gierschik polyclonal                                                                              G.sub.o    Raised vs. pure                                et al PNAS                     holo-protein                                   83:2258, 1986                                                                 Huff et al                                                                              polyclonal                                                                              G.sub.o    Raised vs. pure                                JBC 260:10864                  holo-protein                                   1985                                                                          Tsai et al                                                                              polyclonal                                                                              G.sub.o    Raised vs. pure                                Biochem 26:                                                                             also                 holo-protein                                   4728, 1987                                                                              monoclonal                                                                              TD         Raised vs. TD but                                                             crossreacted G.sub.i.                          Asano et al                                                                             polyclonal                                                                              G.sub.o    Raised vs. pure                                J. Neurochem                   holo-protein                                   48:1617, 1987                                                                 Homburger polyclonal                                                                              G.sub.o    Raised vs. pure                                et al Mol.                     holo-protein                                   Pharm 31:313                                                                  1987                                                                          Katada et al                                                                            polyclonal                                                                              G.sub.o, G.sub.i                                                                         Raised vs. pure holo                           FEBS Letters                   protein. May have                              213:353, 1987                  specificity vs. G.sub.i                        Lerea et al                                                                             polyclonal                                                                              TD rod     Raised vs. synthetic                           Science 234:        TD cone    peptides; specific                             77, 1986                       only for TD but not                                                           for G.sub.i1,2,3.                              Mumby et al                                                                             polyclonal                                                                              TD         Raised vs. pure                                PNAS 83:265                    holo-protein.                                  1986                G.sub.o    Raised vs, pure                                                               holo-protein.                                                      G.sub.o    Raised vs. synthetic                                                          peptide but used an                                                           entirely different                                                            synthetic peptide                                                             than the peptide used                                                         in the present                                                                invention.                                                         Reacts with                                                                              Raised vs. synthetic                                               all G-pro- peptide entirely dif-                                              teins and  ferent from the pep-                                               lacks spec-                                                                              tides employed in the                                              ificity for                                                                              present invention.                                                 any particu-                                                                  lar G-protein.                                            ______________________________________                                    

The decapeptide, KENLKDCGLF, corresponding to the carboxylterminus ofboth rod and cone transducin-alpha includes the cystein residue that isthe site of pertussis toxin-catalyzed ADP-ribosylation. As mentionedabove, the synthetic peptide was conjugated to LKH and three rabbitsdesignated AS/6, 7, and 8 immunized with the peptide KLH conjugate.Preimmune and postimmunization bleeds from each animal were tested forspecific reactivity on immunoblots of purified holotransducin. Forcomparison, an immune bleed of rabbit, AS/1 immunized withholotransducin was also tested. All three peptide immunized rabbitsdeveloped antibodies against transducin-alpha; preimmune sera showed noreactivity. By comparison, AS/1, as previously reported (Gierschik et alsupra. 1985), recognizes all three transducin subunits, alpha, beta andgamma without distinguishing between them.

The carboxyl-terminal decapeptide of transducin-alpha shows somehomology to the carboxyl-terminus of transducin-gamma as well as to aninternal sequence of the "48k" protein of rod outer segments. The latterhomology may reflect the involvement of this domain in receptorinteraction. The reactivity of antisera AS/6 and AS/7 against theserelated sequences was tested by performing immunoblots withholotransducin and the purified 48k protein. AS/6 and AS/7 reactedexclusively with transducin-alpha; AS/1, a holotransducin antiserum,readily recognized transducin-gamma (as well as beta) in this test andboth anti-48k sera tested reacted strongly with this protein (data notshown).

It was of interest to determine if antisera raised against the syntheticpeptide, KENLKDCGLF, could recognize this sequence afterADP-ribosylation on cysteine by pertussis toxin.

To test this, two types of experiments were performed. In the first,intact C6 glioma cells were treated with pertussis toxin, cholera toxin,or no toxin and then the reactivity was tested on immunoblots of AS/7with a membrane preparation from each set of cells. Treatment of intactC6 glioma cells with pertussis toxin abolished subsequentADP-ribosylation of a 40-kDa protein in membranes from treated cellsincubated with pertussis toxin and [alpha-³² P]NAD (not shown). Antiseraraised against KENLKDCGLF, react with 40-41-kDa protein(s) in most cellstested (vide infra) including C6 glioma cells. FIG. 1 shows thereactivity of AS/7 with a 40-Da protein in C6 glioma cell membranes fromcells incubated without toxin or with cholera toxin. In cell membranesfrom cells incubated with pertussis toxin, immunoreactivity is notreduced by the migration of the reactive protein and is slightly reducedas expected after ADP-ribosylation. In a second type of experiment,purified holotransducin was incubated with pertussis toxin for varyingtimes under conditions leading to increasing degrees ofADP-ribosylation. PG/1, an antiserum raised against chemicallyconjugated ADP-ribose

reacts with ADP-ribosylated, but not unmodified, transducin-alpha andshowed an increase in ADP-ribosylationof the transducin-alpha subunitwith increasing time of incubation with pertussis toxin, in agreementwith the results of the experiment shown in FIG. 1. AS/7 reactivity withtransducin-alpha is not affected by pertussis toxin-catalyzedADP-ribosylation (data not shown).

Sequencing of cDNA clones encoding G-protein alpha subunits allowscomparison of the amino acid sequence of the synthetic peptide,KENLKDCGLF, with that predicted by the cDNA clones. Table 4 shows such acomparison and indicates that both G_(i)α-1 and G_(i)α-2 differ from thesynthetic peptide sequence by a single residue. In contrast, G_(o)αdiffers by 5-10 residues. G_(s)α (not shown) shares only 2 residues incommon with the synthetic peptide. Based on this comparison it wasreasoned that antisera raised against KENLKDCGLF might recognizeG_(i)α-1 and G_(i)α-2 subunits, but not G_(o)α.

                  TABLE 4                                                         ______________________________________                                        Comparison of amino acid sequence of syntheitc peptide                        corresponding to transducin-alpha 341-350 to                                  homologous sequences of other G-protein alpha subunits.                       Since the amino sequence of G.sub.o has not been                              published, residue numbers are not shown. (The single                         letter amino acid code is used.)                                              ______________________________________                                        G.sub.iα-1 (345-354):                                                                    K N N L K D C G L F                                          G.sub.iα-2 (346-355):                                                                    K N N L K D C G L F                                          Transducin rod (341-350):                                                                      K E N L K D C G L F                                          Transducin cone (345-354):                                                                     K E N L K D C G L F                                          G.sub.o          A N N L R G C G L Y                                          ______________________________________                                    

To test this hypothesis, immunoblots were performed as shown in FIG. 2.A crude brain membrane preparation or a cholate extract of suchmembranes contained both G_(i) and G_(o). Antisera AS/6 and AS/7revealed an immunoreactive band at 40-41 kDa in cholate extracts ofbrain membranes (FIG. 2, lanes A4 and B4). In contrast, antiserum RV/3raised against a mixture of purified brain G_(i) and G_(o) andpreviously shown to recognize G_(o)α and the common beta subunit reveals39- and 36-kDa immunoreactive bands, as expected (lane 1). By mixingeither AS/6 and RV/3 or AS/7 and RV/3, it was demonstrated (lanes 2 and3) that AS/6 and AS/7 recognizes protein(s), presumably G_(i)α distinctfrom those recognized (G_(o)α and G_(o)β) by RV/3. Identical resultswere obtained with purified G_(i) /G_(o) preparations (not shown).

Antisera raised against KENLKDCGLF thus react with transducin-alpha andG_(i)α but not G_(o)α. These antisera, however, cannot discriminatebetween G_(i)α-1 and G_(I)α-2 which share the identical KNNLKDCGLFsequence (Table 4).

To develope a reagent capable of discriminating between these twoclosely related (88% homologous) sequences, a particular sequence waschosen to prepare antisera capable of differentiating rod and conetransducin-alpha subunits. The sequence of the decapeptide synthesized,LERIAQSDYI, coresponding exactly to that predicted by G_(i)α-2 cDNAscloned from human, rat, and mouse libraries. This sequence is comparedto the homologous sequence of other G-protein alpha subunits, includingbovine and human G_(i)α-1, in Table 5. It should be noted that G_(i)α-1differs in sequence from the synthetic peptide at 3 residues and thatrod and cone transducin-alpha and G_(o)α show further differences.

                  TABLE 5                                                         ______________________________________                                        Comparison of the amino acid sequence of the synthetic                        peptide corresponding to the G.sub.i subunit to                               homologous sequences of other G-potein alph subunits                          The single letter amino acid code is used.                                    ______________________________________                                        Transducin cone (159-168):                                                                     L D R I T A P D Y L                                          Transducin rod (155-164):                                                                      L E R L V T P G Y V                                          G.sub.iα-2 (160-169):                                                                    L E R I A Q S D Y I                                          G.sub.iα-1 (159-168):                                                                    L D R I A Q P N Y I                                          G.sub.oi         L D R I G A A D Y Q                                          ______________________________________                                    

FIG. 3 shows the results of immunoblots of human neutrophil membranes(panel A) and of bovine brain membrane cholate extract (panel B)performed with antisera againt KENLKDCGLF, AS/6 and AS/7, and withantisera LE/1, 2, and 3, raised against a conjugate of the syntheticpepide LERLAQSDYI and carrier protein, KKLH. As reported earlier(Falloon et al, FEBS Letters 209:352-356,1986), AS/6 and AS/7 detect theabundant 40-41-kDa pertussis toxin substrate(s) in neutrophil membranesand in brain. The three preimmune LE sera showed no specific reactivityin either neutrophil membranes or brain cholate extract. All three LEimmune sera recognized a band of similar mobility to that revealed byAS/6 and AS/7 in neutrophil membranes, with LE/1 showing weakerreactivity at equivalent dilution. In brain cholate extract, LE/1 immuneantisera failed to detect specific immunoreactivity, whereas LE/2 andLE/3 revealed bands of similar mobility, but much weaker reactivity, asthose seen with AS/6 and AS/7.

These results indicate that LE antisera detect a protein identical to orclosely realted to G_(i)α-2 that is particularly abundant in neutrophilsand a similar or identical protein in brain that is relatively lower inabundance (compare the ratio of AS/LKE immunoreactivity in neutrophiland brain).

Next the reactivity of LE/3 and AS/6 was compared with purifiedG-protein preparations, including the major pertussis toxin substratepurified from bovine neutrophils. FIG. 4A shows the pattern of proteinstaining of the purified preparations from bovine neutrophil (lane 1,alpha subunit only), bovine brain (lane 2), and bovine rod outersegments (lane 3). Panel B shows the results of immunoblots of theseproteins with three distinct antisera, AS/6, LE/3, and CW/6, a uniqueantiserum raised against holotransducin and shown to cross-react withG_(i)α but not G_(o)α in brain. CW/6 as expected detects the common betasubunit in the two holoprotein preparations, reacts stongly withtransducin-alpha, and with a more slowly migrating (about 41-kDa) G_(i)αfrom brain. CW/6 also cross-reacts with the purified bovine neutrophilprotein, but this reactivity is lower than with the brain protein (notethat 1.0 μg of bovine neutrophil protein was loaded on immunoblot lanescompared with 0.5 μg on the lane stained with Coomassie Blue shown inpanel A). AS/6 reacts equally well with transducin-alpha brain G_(i)αand neutrophil "G_(i)α + as expected, given its ability to recognize theKENLKDCGLF and KNNLKDCGLF sequence. Note the subtle but definitedifferences in migration of the immunoreactive bands detected with A/6.In contrast, as expected (Table 5), LE/3 fails to react with eithertransducin-alpha or G_(o)α. Consistent with the results seen inneutrophil membranes, LE/3 strongly reacts with the purified neutrophilmajor pertussis toxin substrate. Interestingly, LE/3 reveals a faintlyreactive band (lane 2) in the purified brain, G_(I) /G_(o) lane thatcomigrates with the purified neutrophil protein. It is highly likelythat this represents reactivity of LE/3 with a protein similar, if notidenticial, to that in neutrophils, rather than weak cross-reactivitywith the 41-kDa form of G_(i)α abundant in brain that is readilydetected by AS/6. This is based on the clear differences in mobility ofthe bands detected in immunblot (FIG. 4).

The specificity of LE/2 and LE/3 was further assessed by peptidescorresponding to the sequences of rod and cone transducin-alpha subunitsfrom the region homologous to LERIAQSDYI which failed to block LE/2 orLE/3 reactivity with the neutrophil membrane protein. Comparable amounts(1 and 9 μg) of the LERIAQSDYI synthetic peptide efectively blocked LEantisera reactivity (not shown). Also, antisera raised to the syntheticpeptides corresponding to rod and cone transducin-alpha sequences failedto react with the 40-41-kDa protein in neutrophil membranes, althoughthese antisera readily react with rod and cone transducin-alpha.

Tests identical to those illustrated for antisera AS6, AS7, LE/2 andLE/3 have been performed with the other antisera listed in Table 1..These tests likewise demonstrate the specificty and utility of antiseraLD/1, LD/2, GO/1 and GO/3 in discriminating between various G proteins.

From the results presented above, it is quite clear that the antisera ofthe present invention provide means for identification anddifferentiation of various G-proteins.

An antibody reagent kit comprising separate containers each containingdifferent antibodies and optionally different antigens of the presentinvention, makes it possible for the first time either to quantitate ordetect the presence of a specific G-protein in a biological sample. Thisis accomplished by standard immunological techniques employingantibodies or conjugates thereof, well known to one of ordinary skill inthe art. Examples of such techniques are immunoprecipitation, enzymelinked immunosorbent assay, immunofluorescence, immunoblotting,radioimmuno assay and the like. Of course, the antisera or the antibodyreagents can be cryopreserved or lyophilized for prolonged activity.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

What is claimed is:
 1. An antibody which binds specifically to a peptidehaving the amino acid sequence ANNLRGCGLY.
 2. A composition of mattercomprising the antibody of claim 1 and a carrier.
 3. A compostisionaccording to claim 2 wherein the antibody is cryopreserved in a buffer.